RESUMO
Glucagon-like peptide-1 (GLP-1) is a signal peptide released from enteroendocrine cells of the lower intestine. GLP-1 exerts anorectic and antimotility actions that protect the body against nutrient malabsorption. However, little is known about how intestinal GLP-1 affects distant organs despite rapid enzymatic inactivation. We show that intestinal GLP-1 inhibits gastric emptying and eating via intestinofugal neurons, a subclass of myenteric neurons that project to abdominal sympathetic ganglia. Remarkably, cell-specific ablation of intestinofugal neurons eliminated intestinal GLP-1 effects, and their chemical activation functioned as a GLP-1 mimetic. GLP-1 sensing by intestinofugal neurons then engaged a sympatho-gastro-spinal-reticular-hypothalamic pathway that links abnormal stomach distension to craniofacial programs for food rejection. Within this pathway, cell-specific activation of discrete neuronal populations caused systemic GLP-1-like effects. These molecularly identified, delimited enteric circuits may be targeted to ameliorate the abdominal bloating and loss of appetite typical of gastric motility disorders.
Assuntos
Apetite , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Íleo , Neurônios , Estômago , Abdome , Animais , Comunicação Celular , Receptor do Peptídeo Semelhante ao Glucagon 1/metabolismo , Íleo/inervação , Íleo/metabolismo , Masculino , Camundongos , Neurônios/metabolismo , Óxido Nítrico/metabolismo , Transdução de Sinais , Estômago/inervação , Estômago/metabolismoRESUMO
Recent sequencing analyses have shed light on heterogeneous patterns of genomic aberrations in human gastric cancers (GCs). To explore how individual genetic events translate into cancer phenotypes, we established a biological library consisting of genetically engineered gastric organoids carrying various GC mutations and 37 patient-derived organoid lines, including rare genomically stable GCs. Phenotype analyses of GC organoids revealed divergent genetic and epigenetic routes to gain Wnt and R-spondin niche independency. An unbiased phenotype-based genetic screening identified a significant association between CDH1/TP53 compound mutations and the R-spondin independency that was functionally validated by CRISPR-based knockout. Xenografting of GC organoids further established the feasibility of Wnt-targeting therapy for Wnt-dependent GCs. Our results collectively demonstrate that multifaceted genetic abnormalities render human GCs independent of the stem cell niche and highlight the validity of the genotype-phenotype screening strategy in gaining deeper understanding of human cancers.
Assuntos
Adenocarcinoma/patologia , Organoides/patologia , Neoplasias Gástricas/patologia , Estômago/patologia , Trombospondinas/metabolismo , Proteínas Wnt/metabolismo , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Animais , Antígenos CD/genética , Apoptose , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Caderinas/genética , Carcinogênese , Proliferação de Células , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Humanos , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Mutação , Organoides/metabolismo , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismo , Trombospondinas/genética , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/genética , Proteínas Wnt/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Individuals with neurodevelopmental disorders often experience comorbid gastrointestinal distress and dysregulated immune responses, yet the underlying mechanisms remain unclear. In this issue of Immunity, Kim et al. utilize a murine maternal immune activation model of autism and find that inflammation can alter the microbiota of mothers, which postnatally primes offspring CD4+ T cells and increases susceptibility to intestinal inflammation.
Assuntos
Microbioma Gastrointestinal , Microbiota , Efeitos Tardios da Exposição Pré-Natal , Animais , Humanos , Inflamação , Camundongos , EstômagoRESUMO
Neural inputs from internal organs are essential for normal autonomic function. The vagus nerve is a key body-brain connection that monitors the digestive, cardiovascular, and respiratory systems. Within the gastrointestinal tract, vagal sensory neurons detect gut hormones and organ distension. Here, we investigate the molecular diversity of vagal sensory neurons and their roles in sensing gastrointestinal inputs. Genetic approaches allowed targeted investigation of gut-to-brain afferents involved in homeostatic responses to ingested nutrients (GPR65 neurons) and mechanical distension of the stomach and intestine (GLP1R neurons). Optogenetics, in vivo ganglion imaging, and genetically guided anatomical mapping provide direct links between neuron identity, peripheral anatomy, central anatomy, conduction velocity, response properties in vitro and in vivo, and physiological function. These studies clarify the roles of vagal afferents in mediating particular gut hormone responses. Moreover, genetic control over gut-to-brain neurons provides a molecular framework for understanding neural control of gastrointestinal physiology.
Assuntos
Vias Neurais , Neurônios/metabolismo , Células Receptoras Sensoriais/metabolismo , Nervo Vago/metabolismo , Animais , Gânglios/metabolismo , Motilidade Gastrointestinal , Receptor do Peptídeo Semelhante ao Glucagon 1/metabolismo , Camundongos , Optogenética , Receptores Acoplados a Proteínas G/metabolismo , Serotonina/metabolismo , Estômago/inervaçãoRESUMO
Implanted biomaterials and devices face compromised functionality and efficacy in the long term owing to foreign body reactions and subsequent formation of fibrous capsules at the implant-tissue interfaces1-4. Here we demonstrate that an adhesive implant-tissue interface can mitigate fibrous capsule formation in diverse animal models, including rats, mice, humanized mice and pigs, by reducing the level of infiltration of inflammatory cells into the adhesive implant-tissue interface compared to the non-adhesive implant-tissue interface. Histological analysis shows that the adhesive implant-tissue interface does not form observable fibrous capsules on diverse organs, including the abdominal wall, colon, stomach, lung and heart, over 12 weeks in vivo. In vitro protein adsorption, multiplex Luminex assays, quantitative PCR, immunofluorescence analysis and RNA sequencing are additionally carried out to validate the hypothesis. We further demonstrate long-term bidirectional electrical communication enabled by implantable electrodes with an adhesive interface over 12 weeks in a rat model in vivo. These findings may offer a promising strategy for long-term anti-fibrotic implant-tissue interfaces.
Assuntos
Materiais Biocompatíveis , Fibrose , Reação a Corpo Estranho , Próteses e Implantes , Adesivos Teciduais , Animais , Feminino , Humanos , Masculino , Camundongos , Ratos , Parede Abdominal , Adsorção , Materiais Biocompatíveis/química , Colo , Eletrodos Implantados , Fibrose/patologia , Fibrose/prevenção & controle , Reação a Corpo Estranho/prevenção & controle , Reação a Corpo Estranho/patologia , Coração , Pulmão , Camundongos Endogâmicos C57BL , Especificidade de Órgãos , Reação em Cadeia da Polimerase , Ratos Sprague-Dawley , Estômago , Suínos , Fatores de Tempo , Adesivos Teciduais/química , Imunofluorescência , Reprodutibilidade dos Testes , Análise de Sequência de RNARESUMO
Little is known about host-microbiota interactions regulating anti-microbial immunity in the stomach. In this issue, Satoh-Takayama et al. describe an additional immune mechanism involving innate lymphoid cells type 2 (ILC2), which controls infection with Helicobacter pylori, a bacterium associated with inflammation and cancer.
Assuntos
Helicobacter pylori , Linfócitos , Helicobacter pylori/imunologia , Imunidade Inata , Imunoglobulina A , EstômagoRESUMO
The intestinal microbiota shapes and directs immune development locally and systemically, but little is known about whether commensal microbes in the stomach can impact their immunological microenvironment. Here, we report that group 2 innate lymphoid cells (ILC2s) were the predominant ILC subset in the stomach and show that their homeostasis and effector functions were regulated by local commensal communities. Microbes elicited interleukin-7 (IL-7) and IL-33 production in the stomach, which in turn triggered the propagation and activation of ILC2. Stomach ILC2s were also rapidly induced following infection with Helicobacter pylori. ILC2-derived IL-5 resulted in the production of IgA, which coated stomach bacteria in both specific pathogen-free (SPF) and H. pylori-infected mice. Our study thus identifies ILC2-dependent IgA response that is regulated by the commensal microbiota, which is implicated in stomach protection by eliminating IgA-coated bacteria including pathogenic H. pylori.
Assuntos
Microbioma Gastrointestinal/imunologia , Infecções por Helicobacter/imunologia , Helicobacter pylori/patogenicidade , Imunoglobulina A/biossíntese , Interleucina-5/imunologia , Estômago/imunologia , Subpopulações de Linfócitos T/imunologia , Animais , Linhagem da Célula/genética , Linhagem da Célula/imunologia , Feminino , Regulação da Expressão Gênica , Infecções por Helicobacter/microbiologia , Infecções por Helicobacter/patologia , Helicobacter pylori/crescimento & desenvolvimento , Helicobacter pylori/imunologia , Imunidade Humoral , Imunidade Inata , Interleucina-33/genética , Interleucina-33/imunologia , Interleucina-5/genética , Interleucina-7/genética , Interleucina-7/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Cultura Primária de Células , Transdução de Sinais , Estômago/microbiologia , Simbiose/imunologia , Subpopulações de Linfócitos T/classificaçãoRESUMO
The termination of a meal is controlled by dedicated neural circuits in the caudal brainstem. A key challenge is to understand how these circuits transform the sensory signals generated during feeding into dynamic control of behaviour. The caudal nucleus of the solitary tract (cNTS) is the first site in the brain where many meal-related signals are sensed and integrated1-4, but how the cNTS processes ingestive feedback during behaviour is unknown. Here we describe how prolactin-releasing hormone (PRLH) and GCG neurons, two principal cNTS cell types that promote non-aversive satiety, are regulated during ingestion. PRLH neurons showed sustained activation by visceral feedback when nutrients were infused into the stomach, but these sustained responses were substantially reduced during oral consumption. Instead, PRLH neurons shifted to a phasic activity pattern that was time-locked to ingestion and linked to the taste of food. Optogenetic manipulations revealed that PRLH neurons control the duration of seconds-timescale feeding bursts, revealing a mechanism by which orosensory signals feed back to restrain the pace of ingestion. By contrast, GCG neurons were activated by mechanical feedback from the gut, tracked the amount of food consumed and promoted satiety that lasted for tens of minutes. These findings reveal that sequential negative feedback signals from the mouth and gut engage distinct circuits in the caudal brainstem, which in turn control elements of feeding behaviour operating on short and long timescales.
Assuntos
Regulação do Apetite , Tronco Encefálico , Ingestão de Alimentos , Retroalimentação Fisiológica , Alimentos , Saciação , Estômago , Regulação do Apetite/fisiologia , Tronco Encefálico/citologia , Tronco Encefálico/fisiologia , Ingestão de Alimentos/fisiologia , Vias Neurais/citologia , Vias Neurais/fisiologia , Neurônios/metabolismo , Hormônio Liberador de Prolactina/metabolismo , Saciação/fisiologia , Núcleo Solitário/citologia , Núcleo Solitário/fisiologia , Estômago/fisiologia , Paladar/fisiologia , Fatores de Tempo , Animais , CamundongosRESUMO
The physiological functions of mast cells remain largely an enigma. In the context of barrier damage, mast cells are integrated in type 2 immunity and, together with immunoglobulin E (IgE), promote allergic diseases. Allergic symptoms may, however, facilitate expulsion of allergens, toxins and parasites and trigger future antigen avoidance1-3. Here, we show that antigen-specific avoidance behaviour in inbred mice4,5 is critically dependent on mast cells; hence, we identify the immunological sensor cell linking antigen recognition to avoidance behaviour. Avoidance prevented antigen-driven adaptive, innate and mucosal immune activation and inflammation in the stomach and small intestine. Avoidance was IgE dependent, promoted by Th2 cytokines in the immunization phase and by IgE in the execution phase. Mucosal mast cells lining the stomach and small intestine rapidly sensed antigen ingestion. We interrogated potential signalling routes between mast cells and the brain using mutant mice, pharmacological inhibition, neural activity recordings and vagotomy. Inhibition of leukotriene synthesis impaired avoidance, but overall no single pathway interruption completely abrogated avoidance, indicating complex regulation. Collectively, the stage for antigen avoidance is set when adaptive immunity equips mast cells with IgE as a telltale of past immune responses. On subsequent antigen ingestion, mast cells signal termination of antigen intake. Prevention of immunopathology-causing, continuous and futile responses against per se innocuous antigens or of repeated ingestion of toxins through mast-cell-mediated antigen-avoidance behaviour may be an important arm of immunity.
Assuntos
Alérgenos , Aprendizagem da Esquiva , Hipersensibilidade , Mastócitos , Animais , Camundongos , Alérgenos/imunologia , Aprendizagem da Esquiva/fisiologia , Hipersensibilidade/imunologia , Imunoglobulina E/imunologia , Mastócitos/imunologia , Estômago/imunologia , Vagotomia , Imunidade Inata/imunologia , Imunidade nas Mucosas/imunologia , Células Th2/imunologia , Citocinas/imunologia , Leucotrienos/biossíntese , Leucotrienos/imunologia , Intestino Delgado/imunologiaRESUMO
Proliferation of the self-renewing epithelium of the gastric corpus occurs almost exclusively in the isthmus of the glands, from where cells migrate bidirectionally toward pit and base. The isthmus is therefore generally viewed as the stem cell zone. We find that the stem cell marker Troy is expressed at the gland base by a small subpopulation of fully differentiated chief cells. By lineage tracing with a Troy-eGFP-ires-CreERT2 allele, single marked chief cells are shown to generate entirely labeled gastric units over periods of months. This phenomenon accelerates upon tissue damage. Troy(+) chief cells can be cultured to generate long-lived gastric organoids. Troy marks a specific subset of chief cells that display plasticity in that they are capable of replenishing entire gastric units, essentially serving as quiescent "reserve" stem cells. These observations challenge the notion that stem cell hierarchies represent a "one-way street."
Assuntos
Celulas Principais Gástricas/citologia , Células-Tronco/citologia , Estômago/citologia , Animais , Linhagem da Célula , Celulas Principais Gástricas/química , Mucosa Gástrica/citologia , Camundongos , Organoides/citologia , Receptores do Fator de Necrose Tumoral/análise , Via de Sinalização WntRESUMO
The reprogramming factors that induce pluripotency have been identified primarily from embryonic stem cell (ESC)-enriched, pluripotency-associated factors. Here, we report that, during mouse somatic cell reprogramming, pluripotency can be induced with lineage specifiers that are pluripotency rivals to suppress ESC identity, most of which are not enriched in ESCs. We found that OCT4 and SOX2, the core regulators of pluripotency, can be replaced by lineage specifiers that are involved in mesendodermal (ME) specification and in ectodermal (ECT) specification, respectively. OCT4 and its substitutes attenuated the elevated expression of a group of ECT genes, whereas SOX2 and its substitutes curtailed a group of ME genes during reprogramming. Surprisingly, the two counteracting lineage specifiers can synergistically induce pluripotency in the absence of both OCT4 and SOX2. Our study suggests a "seesaw model" in which a balance that is established using pluripotency factors and/or counteracting lineage specifiers can facilitate reprogramming.
Assuntos
Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Fatores de Transcrição/metabolismo , Animais , Células-Tronco Embrionárias/metabolismo , Fibroblastos/metabolismo , Fator de Transcrição GATA3/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/metabolismo , Camundongos , Modelos Biológicos , Fator 3 de Transcrição de Octâmero/metabolismo , Estômago/citologiaRESUMO
Eating and drinking generate sequential mechanosensory signals along the digestive tract. These signals are communicated to the brain for the timely initiation and regulation of diverse ingestive and digestive processes - ranging from appetite control and tactile perception to gut motility, digestive fluid secretion and defecation - that are vital for the proper intake, breakdown and absorption of nutrients and water. Gut mechanosensation has been investigated for over a century as a common pillar of energy, fluid and gastrointestinal homeostasis, and recent discoveries of specific mechanoreceptors, contributing ion channels and the well-defined circuits underlying gut mechanosensation signalling and function have further expanded our understanding of ingestive and digestive processes at the molecular and cellular levels. In this Review, we discuss our current understanding of the generation of mechanosensory signals from the digestive periphery, the neural afferent pathways that relay these signals to the brain and the neural circuit mechanisms that control ingestive and digestive processes, focusing on the four major digestive tract parts: the oral and pharyngeal cavities, oesophagus, stomach and intestines. We also discuss the clinical implications of gut mechanosensation in ingestive and digestive disorders.
Assuntos
Regulação do Apetite , Ingestão de Alimentos , Vias Aferentes/fisiologia , Regulação do Apetite/fisiologia , Trato Gastrointestinal , Humanos , Estômago/fisiologiaRESUMO
R-spondins are critical regulators of gastric epithelial cells, with Lgr5 receptor historically considered as their main signaling transducer. Recent work by Wizenty et al (2022) now revealed distinct roles for Lgr4 and Lgr5 in directing gland reconstitution following H. pylori infection, shedding new light on the complexities of Rspo signaling during gastric regeneration and raising questions about antral stem cell hierarchy.
Assuntos
Receptores Acoplados a Proteínas G , Trombospondinas , Transdução de Sinais , Células-Tronco , Estômago , Trombospondinas/genética , Via de Sinalização WntRESUMO
Helicobacter pylori is a pathogen that colonizes the stomach and causes chronic gastritis. Helicobacter pylori can colonize deep inside gastric glands, triggering increased R-spondin 3 (Rspo3) signaling. This causes an expansion of the "gland base module," which consists of self-renewing stem cells and antimicrobial secretory cells and results in gland hyperplasia. The contribution of Rspo3 receptors Lgr4 and Lgr5 is not well explored. Here, we identified that Lgr4 regulates Lgr5 expression and is required for H. pylori-induced hyperplasia and inflammation, while Lgr5 alone is not. Using conditional knockout mice, we reveal that R-spondin signaling via Lgr4 drives proliferation of stem cells and also induces NF-κB activity in the proliferative stem cells. Upon exposure to H. pylori, the Lgr4-driven NF-κB activation is responsible for the expansion of the gland base module and simultaneously enables chemokine expression in stem cells, resulting in gland hyperplasia and neutrophil recruitment. This demonstrates a connection between R-spondin-Lgr and NF-κB signaling that links epithelial stem cell behavior and inflammatory responses to gland-invading H. pylori.
Assuntos
Helicobacter pylori , Animais , Hiperplasia/metabolismo , Hiperplasia/patologia , Inflamação/patologia , Camundongos , NF-kappa B/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Células-Tronco/metabolismo , EstômagoRESUMO
The stem/progenitor cell pool is indispensable for the development, homeostasis and regeneration of the gastric epithelium, owing to its defining ability to self-renew whilst supplying the various functional epithelial lineages needed to digest food efficiently. A detailed understanding of the intricacies and complexities surrounding the behaviours and roles of these stem cells offers insights, not only into the physiology of gastric epithelial development and maintenance, but also into the pathological consequences following aberrations in stem cell regulation. Here, we provide an insightful synthesis of the existing knowledge on gastric epithelial stem cell biology, including the in vitro and in vivo experimental techniques that have advanced such studies. We highlight the contributions of stem/progenitor cells towards patterning the developing stomach, specification of the differentiated cell lineages and maintenance of the mature epithelium during homeostasis and following injury. Finally, we discuss gaps in our understanding and identify key research areas for future work.
Assuntos
Células-Tronco , Estômago , Homeostase , Diferenciação Celular , Linhagem da CélulaRESUMO
LGR5 marks resident adult epithelial stem cells at the gland base in the mouse pyloric stomach1, but the identity of the equivalent human stem cell population remains unknown owing to a lack of surface markers that facilitate its prospective isolation and validation. In mouse models of intestinal cancer, LGR5+ intestinal stem cells are major sources of cancer following hyperactivation of the WNT pathway2. However, the contribution of pyloric LGR5+ stem cells to gastric cancer following dysregulation of the WNT pathway-a frequent event in gastric cancer in humans3-is unknown. Here we use comparative profiling of LGR5+ stem cell populations along the mouse gastrointestinal tract to identify, and then functionally validate, the membrane protein AQP5 as a marker that enriches for mouse and human adult pyloric stem cells. We show that stem cells within the AQP5+ compartment are a source of WNT-driven, invasive gastric cancer in vivo, using newly generated Aqp5-creERT2 mouse models. Additionally, tumour-resident AQP5+ cells can selectively initiate organoid growth in vitro, which indicates that this population contains potential cancer stem cells. In humans, AQP5 is frequently expressed in primary intestinal and diffuse subtypes of gastric cancer (and in metastases of these subtypes), and often displays altered cellular localization compared with healthy tissue. These newly identified markers and mouse models will be an invaluable resource for deciphering the early formation of gastric cancer, and for isolating and characterizing human-stomach stem cells as a prerequisite for harnessing the regenerative-medicine potential of these cells in the clinic.
Assuntos
Aquaporina 5/metabolismo , Carcinogênese/patologia , Células-Tronco Neoplásicas/patologia , Neoplasias Gástricas/patologia , Estômago/patologia , Animais , Biomarcadores/metabolismo , Humanos , Camundongos , Células-Tronco Neoplásicas/metabolismo , Piloro/patologia , Receptores Acoplados a Proteínas G/metabolismo , Via de Sinalização WntRESUMO
The gastric proton pump (H+,K+-ATPase) transports a proton into the stomach lumen for every K+ ion exchanged in the opposite direction. In the lumen-facing state of the pump (E2), the pump selectively binds K+ despite the presence of a 10-fold higher concentration of Na+. The molecular basis for the ion selectivity of the pump is unknown. Using molecular dynamics simulations, free energy calculations, and Na+ and K+-dependent ATPase activity assays, we demonstrate that the K+ selectivity of the pump depends upon the simultaneous protonation of the acidic residues E343 and E795 in the ion-binding site. We also show that when E936 is protonated, the pump becomes Na+ sensitive. The protonation-mimetic mutant E936Q exhibits weak Na+-activated ATPase activity. A 2.5-Å resolution cryo-EM structure of the E936Q mutant in the K+-occluded E2-Pi form shows, however, no significant structural difference compared with wildtype except less-than-ideal coordination of K+ in the mutant. The selectivity toward a specific ion correlates with a more rigid and less fluctuating ion-binding site. Despite being exposed to a pH of 1, the fundamental principle driving the K+ ion selectivity of H+,K+-ATPase is similar to that of Na+,K+-ATPase: the ionization states of the acidic residues in the ion-binding sites determine ion selectivity. Unlike the Na+,K+-ATPase, however, protonation of an ion-binding glutamate residue (E936) confers Na+ sensitivity.
Assuntos
Simulação de Dinâmica Molecular , Potássio , Potássio/metabolismo , Estômago , Sítios de Ligação , Sódio/metabolismo , Adenosina Trifosfatases/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , ATPase Trocadora de Hidrogênio-Potássio/genética , ATPase Trocadora de Hidrogênio-Potássio/metabolismoRESUMO
There is growing evidence that bacterial morphology is closely related to their lifestyle. The helical Helicobacter pylori relies on its unique shape for survival and efficient colonization of the human stomach. Yet, they have been observed to transform into another distinctive morphology, the spherical coccoid. Despite being hypothesized to be involved in the persistence and transmission of this species, years of effort in deciphering the roles of the coccoid form remain fruitless since contrasting observations regarding its lifestyle were reported. Here, we discuss the two forms of H. pylori with a focus on the coccoid form, the molecular mechanism behind its morphological transformation, and experimental approaches to further develop our understanding of this phenomenon. We also propose a putative mechanism of the coccoid formation in H. pylori through induction of a type-I toxin-antitoxin (TA) system recently shown to influence the morphology of this species.
Assuntos
Infecções por Helicobacter , Helicobacter pylori , Humanos , Helicobacter pylori/genética , Estômago/microbiologia , Infecções por Helicobacter/microbiologiaRESUMO
Mammalian cells synthesize the antioxidant glutathione (GSH) to shield cellular biomolecules from oxidative damage. Certain bacteria, including the gastric pathogen Helicobacter pylori, can perturb host GSH homeostasis. H. pylori infection significantly decreases GSH levels in host tissues, which has been attributed to the accumulation of reactive oxygen species in infected cells. However, the precise mechanism of H. pylori-induced GSH depletion remains unknown, and tools for studying this process during infection are limited. We developed an isotope-tracing approach to quantitatively monitor host-derived GSH in H. pylori-infected cells by mass spectrometry. Using this method, we determined that H. pylori catabolizes reduced GSH from gastric cells using γ-glutamyl transpeptidase (gGT), an enzyme that hydrolyzes GSH to glutamate and cysteinylglycine (Cys-Gly). gGT is an established virulence factor with immunomodulatory properties that is required for H. pylori colonization in vivo. We found that H. pylori internalizes Cys-Gly in a gGT-dependent manner and that Cys-Gly production during H. pylori infection is coupled to the depletion of intracellular GSH from infected cells. Consistent with bacterial catabolism of host GSH, levels of oxidized GSH did not increase during H. pylori infection, and exogenous antioxidants were unable to restore the GSH content of infected cells. Altogether, our results indicate that H. pylori-induced GSH depletion proceeds via an oxidation-independent mechanism driven by the bacterial enzyme gGT, which fortifies bacterial acquisition of nutrients from the host. Additionally, our work establishes a method for tracking the metabolic fate of host-derived GSH during infection.
Assuntos
Infecções por Helicobacter , Helicobacter pylori , Animais , Helicobacter pylori/metabolismo , Infecções por Helicobacter/microbiologia , Estômago , Glutationa/metabolismo , Antioxidantes/metabolismo , Mucosa Gástrica/microbiologia , MamíferosRESUMO
Smooth muscle cells (SMCs), Interstitial cells of Cajal (ICC) and Platelet-derived growth factor receptor α positive (PDGFRα+) cells form an integrated, electrical syncytium within the gastrointestinal (GI) muscular tissues known as the SIP syncytium. Immunohistochemical analysis of gastric corpus muscles showed that c-KIT+/ANO1+ ICC-IM and PDGFRα+ cells were closely apposed to one another in the same anatomical niches. We used intracellular microelectrode recording from corpus muscle bundles to characterize the roles of intramuscular ICC and PDGFRα+ cells in conditioning membrane potentials of gastric muscles. In muscle bundles, that have a relatively higher input impedance than larger muscle strips or sheets, we recorded an ongoing discharge of stochastic fluctuations in membrane potential, previously called unitary potentials or spontaneous transient depolarizations (STDs) and spontaneous transient hyperpolarizations (STHs). We reasoned that STDs should be blocked by antagonists of ANO1, the signature conductance of ICC. Activation of ANO1 has been shown to generate spontaneous transient inward currents (STICs), which are the basis for STDs. Ani9 reduced membrane noise and caused hyperpolarization, but this agent did not block the fluctuations in membrane potential quantitatively. Apamin, an antagonist of small conductance Ca2+-activated K+ channels (SK3), the signature conductance in PDGFRα+ cells, further reduced membrane noise and caused depolarization. Reversing the order of channel antagonists reversed the sequence of depolarization and hyperpolarization. These experiments show that the ongoing discharge of STDs and STHs by ICC and PDGFRα+ cells, respectively, exerts conditioning effects on membrane potentials in the SIP syncytium that would effectively regulate the excitability of SMCs.